TeSaRCoL Stabilization Framework for Microbial Read Count Limits
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Solution Overview
Problem
PCR-based amplification of DNA in biological samples can introduce biases, leading to inaccurate quantification of microbial species, especially for lower-abundant species, and results in high variability between replicates, rendering some applications inoperable.
Innovation Solution
The implementation of Test Sample Read Count Limits (TeSaRCoLs) which involve generating a stabilization framework to determine the viability of stabilization solutions by defining upper and lower read count limits based on the enumerated abundance of microbial species in a control sample, multiplied by sequencing range values.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR-based amplification is used to amplify DNA of microbial species, then the quantity of DNA is increased enabling analysis, but PCR biases are introduced leading to inaccurate quantification especially for lower-abundant species
Solution Approach 1:
The patent introduces sequencing read counts as an intermediary measurement that correlates with microbial abundance without requiring PCR amplification. By using direct sequencing of DNA extracts and establishing empirical relationships between read counts and abundance, the method bypasses PCR biases while still enabling quantification of microbial species including lower-abundant ones
Solution Approach 2:
The patent replaces the mechanical/chemical PCR amplification process with a sequencing-based counting approach. Instead of physically amplifying DNA through enzymatic reactions that introduce biases, the method uses sequencing technology to directly count and enumerate microbial DNA molecules, substituting a measurement-based approach for a manipulation-based approach
2Quantity of substance
If PCR amplification is performed on lower-abundant microbial species, then sufficient DNA quantity is achieved for analysis, but high variability between replicates is introduced rendering applications inoperable
Solution Approach 1:
The patent uses sequencing read counts as a stable intermediary metric that directly reflects microbial abundance without the variability introduced by PCR amplification. By establishing empirical calibration curves between read counts and known abundances, the method achieves reliable and consistent measurements across replicates for lower-abundant species
Solution Approach 2:
The sequencing approach allows each DNA molecule to be independently counted and enumerated, with the data inherently providing its own quantification metric. The read count itself serves as the measurement, eliminating the need for external normalization or correction factors that are required when using PCR-based methods
Data Source
AI summary
Method and systems are provided for defining test sample read count limits for a range of microbial sequence reads. A computing device may generate a stabilization framework usable to determine a viability of a stabilization solution to stabilize a particular microbial species. The stabilization framework may include a first read count limit defined based on an enumerated abundance of the microbial species in a control sample multiplied by a first sequencing range value. The computing device may receive a read count value corresponding to an enumerated abundance of the microbial species in a mixture comprising a biological sample and the stabilization solution. The stabilization framework maybe applied to the read count value to generate an output based on whether the read count value exceeds the first read count limit. The computing device may then select, the stabilizing solution for use with subsequent biological samples.